MDC-15 (ADAM-15, metargidin), a membrane-anchored metalloprotease/disintegrin/cysteine-rich protein, is expressed on the surface of a wide range of cells and has an RGD tripeptide in its disintegrin-like domain. MDC-15 is potentially involved in cell-cell interactions through its interaction with integrins. We expressed a recombinant MDC-15 disintegrin-like domain as a fusion protein with glutathione S-transferase (designated D-15) in bacteria and examined its binding function to integrins using mammalian cells expressing different recombinant integrins. We found that D-15 specifically interacts with ␣v␤3 but not with the other integrins tested (␣2␤1, ␣3␤1, ␣4␤1, ␣5␤1, ␣6␤1, ␣6␤4, ␣v␤1, ␣IIb␤3, and ␣L␤2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to ␣v␤3, suggesting that D-15-␣v␤3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both ␣IIb␤3 and ␣v␤3, suggesting that the receptor binding specificity is mediated by the sequence flanking the RGD tripeptide, as in snake venom disintegrins. These results indicate that the disintegrin-like domain of MDC-15 functions as an adhesion molecule and may be involved n ␣v␤3-mediated cell-cell interactions.Metalloprotease/disintegrin/cysteine-rich proteins (MDCs, also called ADAMs) 1 are membrane-anchored proteins with several domains including a metalloprotease domain, a disintegrin-like domain, a cysteine-rich sequence, an epidermal growth factor-like sequence, a transmembrane domain, and a short cytoplasmic domain (1). The biological functions of MDCs are not clear; however, we do know that fertilins (MDC-1 and -2) (2) are involved in sperm-egg binding and fusion (3), meltrins (MDC-12) (4) are involved in myoblast fusion during muscle development, and KUZ (a Drosophila MDC protein) (5) assists in neurogenesis. The MDC cytoplasmic domain has a proline-rich potential SH3 binding motif, suggesting that MDCcounter receptor interaction may induce signal transduction.Integrins are a family of cell adhesion receptors that bind to a variety of ligands, including extracellular matrix proteins and other cell surface molecules (6 -10). MDCs are potential ligands for integrins, since most snake venom disintegrins interact with integrins ␣IIb␤3 and ␣v␤3 (reviewed in Ref. 11 and references therein). However, little is known about the receptor specificity of MDCs, except that mouse egg integrin ␣6␤1 has been proposed as a receptor for fertilin (2). Evans et al. (12) recently expressed recombinant fertilin fragments in bacteria as fusion proteins with maltose-binding protein (12). The recombinant fertilin-␤ fragment has been shown to bind to the egg membrane to which sperm bind and to block sperm from binding to the egg. These results suggest that the disintegrinlike domains of MDCs may be properly folded in bacteria, that glycosylation of the disintegrin-like domain may not be required for interaction with receptors, and that a strategy using recombinant MDC proteins is a viable alternative to those using puri...

ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.

ADAMs (a disintegrin and metalloproteases) are members of the metzincin superfamily of metalloproteases. Among integrins binding to disintegrin domains of ADAMs are ␣ 9 ␤ 1 and ␣ v ␤ 3 , and they bind in an RGDindependent and an RGD-dependent manner, respectively. Human ADAM15 is the only ADAM with the RGD motif in the disintegrin domain. Thus, both integrin ␣ 9 ␤ 1 and ␣ v ␤ 3 recognize the ADAM15 disintegrin domain. We determined how these integrins recognize the ADAM15 disintegrin domain by mutational analysis. We found that the Arg 481 and the Asp-Leu-Pro-Glu-Phe residues (residues 488 -492) were critical for ␣ 9 ␤ 1 binding, but the RGD motif (residues 484 -486) was not. In contrast, the RGD motif was critical for ␣ v ␤ 3 binding, but the other residues flanking the RGD motif were not. As the RX 6 DLPEF ␣ 9 ␤ 1 recognition motif (residues 481-492) is conserved among ADAMs, except for ADAM10 and 17, we hypothesized that ␣ 9 ␤ 1 may recognize disintegrin domains in all ADAMs except ADAM10 and 17. Indeed we found that ␣ 9 ␤ 1 bound avidly to the disintegrin domains of ADAM1, 2, 3, and 9 but not to the disintegrin domains of ADAM10 and 17. As several ADAMs have been implicated in sperm-oocyte interaction, we tested whether the functional classification of ADAMs, based on specificity for integrin ␣ 9 ␤ 1 , applies to sperm-egg binding. We found that the ADAM2 and 15 disintegrin domains bound to oocytes, but the ADAM17 disintegrin domain did not. Furthermore, the ADAM2 and 15 disintegrin domains effectively blocked binding of sperm to oocytes, but the ADAM17 disintegrin domain did not. These results suggest that oocytes and ␣ 9 ␤ 1 have similar binding specificities for ADAMs and that ␣ 9 ␤ 1 , or a receptor with similar specificity, may be involved in sperm-egg interaction during fertilization. As ␣ 9 ␤ 1 is a receptor for many ADAM disintegrins and ␣ 9 ␤ 1 and ADAMs are widely expressed, ␣ 9 ␤ 1 -ADAM interaction may be of a broad biological importance. ADAMs1 (a disintegrin and metalloproteases) or MDC (metalloprotease/disintegrin/cysteine-rich) proteins are a family of transmembrane glycoproteins of more than 30 members (see www.people.virginia.edu/ϳjag6n/Table_of_the_ADAMs.html and www.gene.ucl.ac.uk/nomenclature/genefamily/metallo. html). ADAMs have a prodomain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGF-like domain, a transmembrane domain, and a cytoplasmic tail (1-3). Several ADAMs are involved in crucial biological processes such as fertilization (ADAM1, 2, and 3) (4 -6) and muscle cell differentiation (meltrin-␣, ADAM12) (7, 8). Most, but not all, ADAMs have a catalytically active metalloprotease domain, which processes several biologically important cell surface proteins including tumor necrosis factor-␣ (tumor necrosis factor converting enzyme (TACE), ADAM17) (for reviews, see Refs. 9 and 10), Alzheimer protein precursor (ADAM10 and 17) (11, 12), Delta (ADAM10) (9, 13, 14), and heparin-binding EGF (ADAM9) (15).The ADAM disintegrin domains are homologous to snake ven...

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.